The effect of paraproteins on the erythrocyte sedimentation rate: a comparison between the StarrSed and TEST 1

This version was published on 1 November 2008

Ann Clin Biochem 2008;45:593-597
doi:10.1258/acb.2008.008062
© 2008 Association for Clinical Biochemistry

 

 

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Original Articles


Maarten T M Raijmakers,
Philip H M Kuijper,
Dirk L Bakkeren and
Huib L Vader


Laboratory of Clinical Chemistry, Máxima Medical Centre, Veldhoven, The Netherlands


Corresponding author: Dr M T M Raijmakers. Email: m.raijmakers{at}mmc.nl




Abstract

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Background: The principle of the erythrocyte sedimentation rate (ESR) asassessed by TEST 1 is different from that of Westergren-basedmethods. This could result in different influences on the testsby paraproteins.

Methods: We investigated the effect of paraproteins on ESR readings by TEST 1 (y) and the StarrSed (x), a Westergren-based method,in 142 patients with paraproteinaemia. Agreement (Passing-Bablok)and bias (Bland–Altman) between methods was investigatedand compared with that of a control population.

Results: A poor agreement between the two methods was found in patients with a paraprotein (y = 0.67x + 3.3) in comparison with that of the control population (y = 0.96x + 0.2). Large differencesbetween methods were present when ESR readings were >40 mm/hour,but clinical interpretation was similar in 90% of cases. Linearregression showed a concentration dependent influence of paraproteinson ESR readings by the StarrSed, especially for immunoglobulinclass IgM.

Conclusion: ESR readings by TEST 1 result in similar clinical interpretationfor most subjects, but readings are less influenced by the presenceof a paraprotein than those of a Westergren-based method.





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Measurement of the erythrocyte sedimentation rate (ESR), also known as the length of sedimentation reaction in blood, is an easy and inexpensive laboratory technique that has been used as a non-specific screening procedure to assess the acute-phase response for many years.1 Determination of the ESR is also commonly used for monitoring of disease activity in rheumatoid arthritis as it is part of the Disease Activity Score in 28 joints.2 The International Council for Standardization in Haematology has recommended the original method described by Westergren as the gold standard.1 The Westergren method measures the plasma column after one hour of spontaneous sedimentation. The ESR reading is thus a representation of the physical process of erythrocyte sedimentation that can schematically be divided into three phases. After the lag-phase in which an initial reorientation of the individual erythrocytes takes place, an erythrocyte sediment is formed mainly under influence of gravitational forces and formation of erythrocyte aggregates that are formed after shielding the negative zeta-potential. In the last phase, the erythrocyte sediment is more tightly packed, which has a minimal influence on the final ESR result. Any condition that is associated with increased concentrations of positively charged molecules will lead to an elevated ESR reading. Examples of such molecules are the proteins of the acute-phase response (e.g. fibrinogen) and immunoglobulins. In particular, paraproteins (i.e. a monoclonal expression of an immunoglobulin) that can be found in multiple myeloma or Waldenstrom’s macroglobulinaemia will enhance rouleaux formation, resulting in increased ESR readings.35

All techniques that are based on the Westergren principle have the major disadvantages of being time-consuming. In the TEST 1 (Alifax, Padovo, Italy), ESR is assessed using a quantitative capillary photometry-based technology.6 After three minutes of mixing, the erythrocyte microsedimentation is measured at 37°C by centrifuging each sample at about 20xg and simultaneouslytaking 1000 readings (950 nm) during the first 20 seconds ofsedimentation. From these readings, a sedimentation curve isplotted, and the data are converted into Westergren values bymeans of a linear regression model. After starting a new batchof samples, the TEST 1 will print the first result after threeminutes, 20 seconds and consecutive results will be printedwith 20-second intervals.

Previous studies performed in subjects of the general population have shown a good correlation between ESR results by TEST 1 and Westergren-based methods.79 These studies have nottaken into account the influence of the presence of paraproteinson ESR readings. As the TEST 1 measures the ESR in the initiallag-phase, we hypothesized that this method may be less influencedby the presence of a paraprotein compared with other methodsbased on the Westergren principle. The aim of this study was,therefore, to investigate the effect of paraproteins on theESR measured with the TEST 1 in comparison with the ESR measuredwith the StarrSed, a Westergren-based method.





Materials and methods

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Patient collection and methods

From February until October 2007 142 consecutive patients, known to have a paraprotein, and who were visiting the laboratory for routine paraprotein concentration monitoring, were asked to participate in the study. Informed consent was obtained from each patient before one additional tube containing K3-ethylenediaminetetraaceticacid was collected to be able to simultaneously asses the ESRusing the TEST 1 and the StarrSed (Goffin Meyvis, Etten-Leur,The Netherlands), which is a Westergren-based method. In eachpatient, the type and the concentration of the paraprotein wasdetermined by electrophoresis followed by immunofixation onthe Sebia Hydrasys electrophoresis system using agarose gelsand the Hydragel 6/12 IF Pentakit (SEBIA Benelux N.V., Issy-les-Moulineaux,France). Data of 102 consecutive hospital patients from a validationprotocol that was previously performed by the authors (datanot published) served as a control population. In this validationprotocol, ESR was similarly measured using the same techniquesas in the present study. To rule out infection or other inflammatoryprocesses C-reactive protein (CRP) was measured using an immunoturbidimetricassay on the Roche Modular P-Module (Roche Diagnostics GmbH,Mannheim, Germany) in combination with a leukocyte count thatwas measured using the LH750 (Beckman-Coulter Nederland BV,Mijdrecht, The Netherlands).


Statistics

Data were analysed using MS Excel 2003 software (MicrosoftTM, Redmond, WA, USA) and Analyse-It v1.72 (Analyse-It Software Ltd., Leeds, UK). Passing-Bablok analysis was used to compare ESR values, and Bland–Altman analysis was used to evaluate bias and 95% CI limits of agreement. Differences between methods were tested using the Wilcoxon-signed rank tests. Linear regression was used to investigate the effect of paraprotein concentration on measured ESR. For the linear regression analysis samples in which paraprotein concentration could not be determined quantitatively from the electrophoresis pattern were excluded. Values of P< 0.05 were considered to be statistically significant.





Results

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In the hospital population, a good agreement between the StarrSed (x) and the TEST 1 (y) was found with a regression equation of the Passing-Bablok method comparison of y = 0.96x + 0.17 (95% confidence interval [CI] slope 0.85–1.05 and intercept –0.84 to 1.52, Figure 1). In contrast, the presence of a paraprotein (concentration up to 55 g/L) resulted in poorer agreement between these methods y = 0.67x + 3.33 (95% CI slope 0.57–0.77 and intercept 0.46–4.29). When ESR values <40 mm/hour only were included, a good agreement with a regression equation of y = 1.00x + 0.00 (95% CI slope 0.82–1.31 andintercept –3.31 to 1.68) was found. From the Bland–Altmanplots, it becomes clear that the poor agreement between bothmethods arises from the large divergence when higher ESR valuesare measured. ESR readings of >40 mm/hour show not only alarger difference between both methods, but individual ESR readingsof the StarrSed are also higher than those of the TEST 1. Thisresults in a larger mean bias in patients with a paraprotein(–12.5 mm/hour, 95% CI –16.9 to –8.0 mm/hour)when compared with that of a random hospital population (–0.9mm/hour, 95% CI –2.4 to 0.6 mm/hour).



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Figure 1 Comparison of erythrocyte sedimentation rate measured with the StarrSedd and TEST 1 in a hospital population (a/c) and in patients with a paraprotein (b/d). (a and b) Passing-Bablok plots. Regression equations are shown in the graph. (c and d) Bias plots. Horizontal lines (– • –) denotes 95% limits of agreement (c: –15.8 to 14.1 mm/hour and d: –65.1 to 40.1 mm/hour)

 

Owing to the molecular structure of paraprotein subtypes, it may be expected that different classes behave differently in ESR measurement. Therefore, a subgroup analysis, in which three groups were created according to immunoglobulin class (IgG (n = 86; concentration up to 55 g/L), IgA (n = 21; concentration up to 47 g/L) or IgM (n = 35; concentration up to 43 g/L)), was performed. Paraproteins of class IgG and IgA showed a poor agreement between the two methods with a slope of the Passing-Bablok curve similar for both paraprotein classes (Table 1). The agreement between methods seemed to be even worse for paraproteins of class IgM as the slope of the Passing-Bablok curve was lower, but with an overlap of the 95% CIs. The large difference in ESR readings for ESR readings >40 mm/hour contributed to these poor agreements, which was also reflected in the bias plot per paraprotein class. The difference in ESR readings between the StarrSed and the TEST 1 was significantly different for paraprotein subclasses IgG and IgM. However, as a large number of patients with paraprotein class IgG (n = 63; 73%) had anESR reading <40 mm/hour which influenced mean bias and 95%limits of agreement, the mean bias and 95% limits of agreementfor this class were the smallest. The largest difference betweenmethods was found for paraprotein of class IgM.



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Table 1 Relationship of erythrocyte sedimentation rate readings by TEST 1 (y) to StarrSed (x) for paraprotein classes

 

The clinical impact of both methods was investigated by evaluating the effect on clinical interpretation (i.e. ESR below or above the reference level for both methods) and by comparing absolute bias. Although TEST 1 ESR readings were on average lower than those of the StarrSed, a good concordance with respect to clinical interpretation was found (Table 2). More importantly, discordantresults were only found when ESR reading were low (highest discordantESR reading for the StarrSed was 30 mm/hour) in combinationwith low paraprotein concentrations (mean concentration of 4.6g/L for IgG and 2.1 g/L for IgM). In addition, for paraproteinsof class IgG and IgA individual ESR readings by TEST 1 werelower in around 50% of the cases, indicating that some patientswill have a lower ESR reading when the TEST 1 is used insteadof a Westergren-based method, but that an equal population ofpatients will have a higher ESR reading in comparison with thatof a Westergren-based method. Thus both methods will show elevatedESR readings in an equal proportion, but a different subset,of patients with undiagnosed paraprotein of class IgG or IgA.Unfortunately, this did not seem to be valid for patients witha paraprotein of class IgM. Overall, these findings indicatethat clinical decision will be relatively uninfluenced by themethod used for ESR measurement. In the majority of cases whenusing TEST 1 the presence of a paraprotein results in an ESRreading above the reference value, but this increase is lesspronounced in comparison with an ESR reading by the StarrSed.



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Table 2 Evaluation of clinical impact TEST 1 versus StarrSed

 

We hypothesized that the observed differences were dependent on the amount of paraprotein present. Therefore, the relationship between paraprotein concentration and height of ESR reading was investigated using linear regression. For the overall population under study, it was found that both methods were influenced by the presence of a paraprotein (Table 3). However, ESRreadings by the StarrSed were more influenced than those measuredby the TEST 1, with the slope of the linear regression equationbeing 2.6 times higher (2.4 versus 0.9). As the 95% CIs do notoverlap this difference can be considered statistically different.This implies that for each gram per litre paraprotein, the StarrSedreading is increased by 2.4 mm/hour compared with 0.9 mm/hourfor the TEST 1. For the StarrSed, a significant linear relationshipwas found for the presence of a paraprotein of either immunoglobulinclass. The largest effect was found for paraproteins of classIgM followed by IgA. Surprisingly, a significant correlationwas only found between the ESR reading by TEST 1 and paraproteinof class IgG, but not for those of class IgA and IgM. AlthoughESR readings on both methods were dependent on the amount ofparaprotein of class IgG, the slopes of the regression linefor the StarrSed was approximately two-fold higher. It can beconcluded that the presence of a paraprotein has a larger quantitativeeffect on ESR readings measured by the StarrSed than by TEST1. Within ESR measurements on the StarrSed, the effect of paraproteinsof class IgM and IgA is larger than that of class IgG.



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Table 3 Relationship between paraprotein concentration (x) and erythrocyte sedimentation rate readings (y) by TEST 1 and the StarrSed using linear regression





Discussion

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We have shown that among patients with a paraprotein, thereis a negligible difference in clinical interpretation when ESRis measured with either the StarrSed or the TEST 1. However,the presence of a paraprotein has different impact on the ESRreading. In particular, when ESR readings exceeded 40 mm/houra large difference between the methods was observed. This canbe explained by the paraprotein dependency of the ESR reading,which is more pronounced in the StarrSed in comparison withTEST 1 method.

This is the second study known to us that has investigated the influence of paraproteins measured by TEST 1 in comparison with a Westergren-based method. In a previous study, Ajubi et al.10 have shown a linear relationship between ESR reading by TEST 1 (x) and paraprotein concentration (y) (y = 0.22x + 0.69; r = 0.71). These findings correspond well with those of our study when they are presented with ESR put on the x-axis (y = 0.17x + 5.5; r = 0.39). However, their finding that such a relationship is also present in a subgroup of patients with a paraprotein of class IgM (y = 0.23x – 0.95; r = 0.93) are in contradiction to our finding, because we did not find a correlation between ESR reading and the concentration of paraprotein of class IgM (y = 0.08x + 6.9; r = 0.20). This discrepancy could possibly be explained by the differences in composition of the investigated groups. Whereas the study of Ajubi et al.10 consisted of few subjects with paraprotein of class IgM (n = 9) with concentrations up to 20 g/L, our study was larger (n = 28) with paraproteinconcentrations up to 43 g/L.

In addition to the study of Ajubi et al.10 we have also investigatedthe relationship between paraprotein concentrations of all threeclasses for both the TEST 1 and the StarrSed. Our results indicatethat ESR values measured by the StarrSed are paraprotein concentrationdependent, whereas those measured by TEST 1 are not with theexception of paraprotein of class IgG. For the overall studypopulation, the influence of ESR readings by the StarrSed inthe presence of a paraprotein was statistically greater thanthat measured by the TEST 1. The different influence of paraproteinson ESR readings in both methods might be explained by the dissimilaritiesin method principles. As described earlier, erythrocyte sedimentationoccurs in three phases. In TEST 1, the ESR readings take placein the lag-phase before the actual sedimentation of the erythrocytes.In this phase only small changes in erythrocyte distributionoccur as reorientation of erythrocytes takes place. As thisprocess is most probably not influenced by the presence of aparaprotein, there will only be a minimal effect on the ESRreading. In contrast, in Westergren-based methods the ESR readingsare largely influenced by the physical interactions in the secondphase of the process. As paraprotein of class IgM has the greatesteffect, the interaction of immunoglobulins with the negativelycharged erythrocytes is most likely size-dependent. Moreover,as a pentamer, IgM has not only the best capability to shieldthe negative charges on the surface of an erythrocyte, but itssize will also enhance rouleaux formation by coupling individualerythrocytes. This also explains why different regression slopeswere found for different paraprotein classes.

As the influence of paraproteins on the TEST 1 ESR result is minimal in comparison with that of the StarrSed it may be postulated that TEST 1 is a better method to use as a screening technique for the presence of an infection in patients with a paraprotein. During the study, we have also collected data on CRP and leukocyte counts, but the majority of the subjects (n = 111; 78%) hada CRP concentration <6 mg/L and only two of the subjectswith an increased CRP concentration had a leukocytosis indicatingthat most of the sampled patients did not show any signs ofinfection during this routine follow-up. Therefore, we werenot able to investigate this hypothesis and a new study shouldbe designed to do so.

In conclusion, this study supports the validity of TEST 1 forESR measurement in patients with a paraprotein. ESR readingsby TEST 1 result in similar clinical interpretation, but areless affected by the presence of a paraprotein, compared withWestergren-based methods. Conversely, TEST 1 is, therefore,not a useful indicator of the presence of a paraprotein.

 

 

 

 




ACKNOWLEDGEMENTS

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The authors would like to thank Arno I W de Natris for his excellenttechnical assistance during the study period.

(Accepted June 24, 2008)



REFERENCES

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 REFERENCES


  1. International Council for Standardization in Haematology (Expert Panel on Blood Rheology). ICSH recommendations for measurement of erythrocyte sedimentation rate. J Clin Pathol 1993;46:198–203[Abstract/Free Full Text]
  2. Fransen J, van Riel PL. DAS remission cut points. Clin Exp Rheumatol 2006;24:S29–32
  3. Cesana C, Klersy C, Barbarano L, et al. Prognostic factors for malignant transformation in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. J Clin Oncol 2002;20:1625–34[Abstract/Free Full Text]
  4. Morra E, Cesana C, Klersy C, et al. Clinical characteristics and factors predicting evolution of asymptomatic IgM monoclonal gammopathies and IgM-related disorders. Leukemia 2004;18:1512–7[Medline]
  5. Gore ME, Riches PG, Kohn J. Identification of the paraproteins and clinical significance of more than one paraprotein and clinical significance of more than one paraprotein in serum of 56 patients. J Clin Pathol 1979;32:313–7[Abstract/Free Full Text]
  6. Plebani M, De TS, Sanzari MC, Bernardi D, Stockreiter E. The TEST 1 automated system: a new method for measuring the erythrocyte sedimentation rate. Am J Clin Pathol 1998;110:334–40[Medline]
  7. Romero A, Munoz M, Ramirez G. Length of sedimentation reaction in blood: a comparison of the test 1 ESR system with the ICSH reference method and the sedisystem 15. Clin Chem Lab Med 2003;41:232–7[Medline]
  8. Ozdem S, Akbas HS, Donmez L, Gultekin M. Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood. Clin Chem Lab Med 2006;44:407–12[Medline]
  9. de JN, Sewkaransing I, Slinger J, Rijsdijk JJM. Erythrocyte sedimentation rate by the test-1 analyzer. Clin Chem 2000;46:881–2[Free Full Text]
  10. Ajubi NE, Bakker AJ, van den Berg GA. Determination of the length of sedimentation reaction in blood using the TEST 1 system: comparison with the Sedimatic 100 method, turbidimetric fibrinogen levels, and the influence of M-proteins. Clin Chem Lab Med 2006;44:904–6[Medline]


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